1. Field of the Invention
The present invention is broadly concerned with improved enzyme preparations which are capable of catalyzing a variety of chemical reactions carried out in organic solvents. More particularly, the invention is concerned with such enzyme preparations, methods of preparing the preparations, and use thereof in catalyzed reactions, wherein the enzyme preparations comprise enzyme molecules electrostatically adhered to nanoscale solid particles such as fumed silica or fumed alumina.
2. Description of the Prior Art
So-called “nonaqueous enzymology” has become an important area of research and development. Enzymes exhibit a wide array of novel reactivities and selectivities in non-aqueous solvents. For example, many reactions that are impossible in water due to kinetic or thermodynamic reasons can be performed in organic solvents due to the suppression of water-induced side reactions. Improved and altered substrate specificities and selectivities can be observed. Examples of practical applications are enantioselective synthesis and combinatorial biocatalysis. The possibility of the solubilization of hydrophobic substrates or products in organic solvents opens opportunities for the enzymatic production of poorly water soluble fine chemicals and pharmaceuticals. The thermal and storage stability of enzymes can be significantly enhanced in nonaqueous media.
One challenge for the use of enzymes in organic media is the decreased catalytic activity which is in general orders of magnitude lower compared to aqueous solution. Several methods to overcome this disadvantage have been investigated. Reversed micelles, the immobilization on a variety of materials, or surface modification of the enzyme can be employed. Immobilization on ceramics was reported for the improvement of enzymatic catalysis at low temperatures. Addition of disodium hydrogenphosphate or L-alanine prior to lyophilization was reported to improve enantioselectivity but not the enzyme activity over native lipase for a reaction in isopropylether.
One of the most successful methods to increase the relatively low activity of enzymes in organic media is the addition of inorganic salts before lyophilization of the enzyme. The activity of subtilisin Carlsberg (SC) in different organic solvents was increased almost 4000 fold by addition of 98 wt % of KCl (relative to the final enzyme preparation mass) to the enzyme in aqueous buffer solution prior to lyophilization. At optimum water concentration in the solvent a 27000 fold enhanced activity of SC in hexane compared to the salt-free lyophilized enzyme was reported. (Ru et al., Biotechnol. Bioeng. 75 (2001) 187, hereafter “Ru et al.”) See also U.S. Pat. No. 6,171,813.
The detailed mechanism of this enzyme activation by freeze-drying in presence of KCl is not entirely clear. It has been hypothesized that the enzyme-bound water (sometimes termed essential water) is the main factor of altered enzyme activity in organic media. This water is thought to provide the enzyme molecule with the internal mobility which is apparently necessary for enzymatic catalysis. By adding highly kosmotropic salts prior to freeze-drying aqueous enzyme solutions this essential water is thought to be supplied to the enzyme via the salt. According to this view, the presence and type of salt would have a significant influence on activation.
However, salt-activation of enzymes for use in organic solvents presents a number of practical difficulties. The preparative procedure is relatively cumbersome, requiring pre-freezing in liquid nitrogen to achieve high surface areas for enzyme immobilization. Moreover, no simple particle size control is possible, and it is very difficult to adapt the procedure to pound-scale production owing to the intricate salt crystallization process. Finally, some of the proposed salts and salt mixtures are relatively expensive (e.g., CsCl, on the order of $150.00/pound), and the natural hygroscopicity of salts present storage and fabrication problems.